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Abstract:

The present invention relates to an AN3-based protein complex. It relates
further to the use of the complex to promote plant growth, and to a
method for stimulating the complex formation, by overexpressing at least
two member of the complex.

2. An isolated AN3-based protein complex comprising at least the proteins
AN3p and one or more proteins selected from the group consisting of ARP4
(AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C
(AT1G21700) and SWP73B (AT5G14170).

3. The isolated AN3-based protein complex of claim 2, wherein said
protein complex comprises at least AN3p, an actin related protein
selected from the group consisting of ARP4 and ARP7, an ATPase selected
from the group consisting of SNF2 (BRM) and SYD, and a SWIRM domain
containing protein.

5. A method of promoting plant growth comprising simultaneously
overexpressing at least two proteins of the complex of claim 1.

6. A method to promote AN3-based protein complex formation comprising
simultaneously ovexpressing at least two proteins of the complex.

Description:

[0001] The present invention relates to an AN3-based protein complex. It
relates further to the use of the complex to promote plant growth, and to
a method for stimulating the complex formation, by overexpressing at
least two members of the complex.

[0002] The demand for more plant derived products has spectacularly
increased. In the near future the challenge for agriculture will be to
fulfill the growing demands for feed and food in a sustainable manner.
Moreover plants start to play an important role as energy sources. To
cope with these major challenges, a profound increase in plant yield will
have to be achieved. Biomass production is a multi-factorial system in
which a plethora of processes are fed into the activity of meristems that
give rise to new cells, tissues, and organs. Although a considerable
amount of research on yield performance is being performed little is
known about the molecular networks underpinning yield (Van Camp, 2005).
Many genes have been described in Arabidopsis thaliana that, when mutated
or ectopically expressed, result in the formation of larger structures,
such as leaves or roots. These so-called "intrinsic yield genes" are
involved in many different processes whose interrelationship is mostly
unknown.

[0003] One of these "intrinsic yield genes", AN3 (also known as GIF1), was
identified in search of GRF (growth regulating factor) interactors (Kim
and Kende, 2004) and by analysis of narrow-leaf Arabidopsis mutants
(Horiguchi et al., 2005). AN3 is a homolog of the human SYT (synovial
sarcoma translocation) protein and is encoded by a small gene family in
the Arabidopsis genome. SYT is a transcription co-activator whose
biological function, despite the implication of its chromosomal
translocation in tumorigenesis, is still unclear (Clark et al., 1994; de
Bruijn et al., 1996). Using the yeast GAL4 system, AN3 was shown to
possess transactivation activity (Kim and Kende, 2004). This together
with yeast two-hybrid and in vitro binding assays demonstrating
interaction of AN3 with several GRFs (Kim and Kende, 2004; Horiguchi et
al., 2005), suggests a role of AN3 as transcription co-activator of GRFs.
GRF (growth regulating factor) genes occur in the genomes of all seed
plants thus far examined and encode putative transcription factors that
play a regulatory role in growth and development of leaves (Kim et al.,
2003). In support of a GRF and AN3 transcription activator and
co-activator complex, grf and an3 mutants display similar phenotypes, and
combinations of grf and an3 mutations showed a cooperative effect (Kim
and Kende, 2004). The an3 mutant narrow-leaf phenotype is shown to result
of a reduction in cell numbers. Moreover, ectopic expression of AN3
resulted in transgenic plants with larger leaves consisting of more
cells, indicating that AN3 controls both cell number and organ size
(Horiguchi et al., 2005). Although the function of AN3 in plant growth
regulation is not known, these results show that AN3 fulfills the
requirements of an "intrinsic yield gene".

[0004] In our ambition to decipher the molecular network underpinning
yield enhancement mechanism a genome-wide protein centered approach was
undertaken to study AN3 interacting proteins in Arabidopsis thaliana cell
suspension cultures. The tandem affinity purification (TAP) technology
combined with mass spectrometry (MS) based protein identification
resulted in the isolation and identification of 25 AN3 interacting
proteins that may function in the regulation of plant growth (Table 2).
Surprisingly, we isolated several proteins belonging to multiprotein
complexes. Moreover, many interactors are completely uncharacterized.
Reports on few of the AN3 interactors show that they are implicated in
several developmental processes (Wagner & Meyerowitz, 2002; Meagher et
al., 2005; Sarnowski et al., 2005; Hurtado et al., 2006; Kwon et al.,
2006) but so far none of the identified genes have been associated with
stimulation of plant growth.

[0005] A first aspect of the invention is an isolated AN3-based protein
complex, comprising at least the proteins AN3p and one or more of the
proteins selected from the group encoded by AT4G16143, AT1G09270,
AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020, AT2G28290,
AT1G21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980, AT5G55210,
AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030, AT2G 18876,
AT5G17510, AT1G05370, AT4G21540, AT1G23900 and AT5G23690 (genes listed in
Table II). Preferably, said AN3-based protein complex comprises at least
the proteins AN3p and one or more proteins selected from the group
consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD
(AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170). Even more
preferably, said AN3-based protein complex comprises at least AN3p, an
actin related protein selected from the group consisting of ARP4 and
ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD
and a SWIRM domain containing protein. Preferably, said SWIRM domain
containing protein is SWI3C. An AN3-based protein complex as used here
means that AN3p is interacting, directly or indirectly, with the other
proteins of the complex. A direct interaction is an interaction where at
least one domain of AN3p interacts with one or more domains or the
interaction partner. An indirect interaction is an interaction where AN3p
itself is not interacting with the interacting protein by one of its
domains, but where said interacting protein is interacting with a protein
that is directly or indirectly interacting with AN3p.

[0006] A further aspect of the invention is the use of a protein complex
according to the invention to promote plant growth. Preferably, said use
is an overexpression of the protein complex, by overexpressing at least
two members of the protein complex. Promotion of plant growth, as used
here, is an increase in plant biomass in plants where the protein complex
is used, compared with the same plant where the complex is not used,
grown under the same conditions, except for the conditions needed for the
use of the complex, if any. Such conditions may be, as a non limited
example, the addition of one or more compounds to induce one or more
promoters of one or more genes encoding a protein of the complex.
Alternatively, the same plant is an untransformed parental plant, grown
under the same conditions as the transformed plant, wherein the complex
is used. Preferably, promotion of plant growth results in an increased
yield. This yield can be a total increase in plant biomass, or a partial
increase of yield, such as, but not limited to seed yield, leave yield or
root yield.

[0007] Still another aspect of the invention is a method to promote
AN3-based protein complex formation, by simultaneous overexpression of at
least two proteins of the complex. Proteins of the complex, beside AN3p
itself, are listed in table II. Preferably, said overexpression is an
overexpression of AN3p and one or more proteins selected from the group
consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD
(AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170). Even more
preferably, said overexpression is an overexpression of at least AN3p, an
actin related protein selected from the group consisting of ARP4 and
ARP7, an ATPase selected from the group consisting of SNF2 (BRM) and SYD
and a SWIRM domain containing protein. Preferably, said SWIRM domain
containing protein is SWI3C.

[0008] Methods for obtaining overexpression are known to the person
skilled in the art, and comprise, but are not limited to placing the gene
encoding the protein to be overexpressed after a strong promoter such as
the Cauliflower Mosaic Virus 35S promoter. Simultaneous overexpression as
used here means that there is an overlap in timeframe for all the
proteins to be overexpressed, whereby the level of said proteins is
increased when compared to a non-overexpressed control. It does not
necessarily mean that all genes should be induced at the same moment.
Depending upon the turnover of the messenger RNA and/or the protein, one
gene may be induced before or after another, as long as there is an
overlap in time where both proteins are present in a concentration that
is higher than the normal (non-overexpressed) concentration.

[0010] The total protein extract of 2-day-old wild-type and N- and
C-terminal GS-tagged GFP and AN3 overexpressing cultures (60 μg) was
separated by 12% SDS-PAGE and immunoblotted. For detection of GS-tagged
proteins, blots were incubated with human blood plasma followed by
incubation with anti-human IgG coupled to horseradish peroxidase. Protein
gel blots were developed by Chemiluminiscent detection. The expected
recombinant molecular masses for GS-tagged GFP and AN3 are 52.8 kDa and
43.5 kDa, respectively (indicated with a black dot).

[0013] Construction of N- and C-terminal GS-tagged GFP and AN3 under the
control of the 35S (CaMV) promoter was obtained by Multisite Gateway LR
reactions. The coding regions, without (-) and with (+) stopcodon, were
amplified by polymerase chain reaction (PCR) and cloned into the Gateway
pDONR221 vector (Invitrogen) resulting in pEntryL1L2-GFP(-)
pEntryL1L2-GFP(+), pEntryL1L2-AN3(-) and pEntryL1L2-AN3(+). The
Pro35S:GFP-GS- and Pro35S:AN3-GS-containing plant
transformation vectors were obtained by Multisite Gateway LR reaction
between pEntryL4R1-Pro35S, pEntryL1L2-GFP(-) or pEntryL1L2-AN3(-),
and pEntryR2L3-GS and the destination vector pKCTAP, respectively (Van
Leene et al., 2007). To obtain the Pro35S:GS-GFP and Pro35S:GS-AN3
vectors Multisite LR recombination between pEntryL4L3-Pro35S and
pEntryL1L2-GFP(+) or pEntryL1L2-AN3(+) with pKNGSTAP occurred.

[0016] The Arabidopsis culture was transformed by Agrobacterium
co-cultivation as described previously (Van Leene et al., 2007). The
Agrobacterium culture exponentially growing in YEB (OD600 between
1.0 and 1.5) was washed three times by centrifugation (10 min at 5000
rpm) with an equal volume MSMO medium and resuspended in cell suspension
growing medium until an OD600 of 1.0. Two days after subcultivation,
3 mL suspension culture was incubated with 200 μL washed Agrobacteria
and 200 μM acetoseringone, for 48 h in the dark at 25° C. with
gentle agitation (130 rpm). Two days after co-cultivation, 7 mL MSMO
containing a mix of three antibiotics (25 μg/mL kanamycin, 500
μg/mL carbenicellin, and 500 μg/mL vancomycin) was added to the
cell cultures and grown further in suspension under standard conditions
(25° C., 130 rpm and continuous darkness). The stable transgenic
cultures were selected by sequentional dilution in a 1:5 and 1:10 ratio
in 50 mL fresh MSMO medium containing the antibiotics mix, respectively
at 11, and 18 days post co-cultivation. After counter selecting the
bacteria, the transgenic plant cells were further subcultured weekly in a
1:5 ratio in 50 mL MSMO medium containing 25 μg/mL kanamycin for two
more weeks. Thereafter the cells were weekly subcultured in fresh medium
at a 1/10 dilution.

[0025] In order to identify the interaction partners of AN3 in vivo, we
performed tandem affinity (TAP) purifications on N- and C-terminal
GS-fusions of AN3 ectopically expressed under control of the constitutive
35SCaMV promoter in transgenic Arabidopsis suspension cultures. Two
independent TAP purifications were performed on extracts from AN3-GS and
GS-AN3 lines, harvested two days after sub-culturing into fresh medium.
The affinity purified proteins were separated on a 4-12% NuPAGE gel and
stained with Coomassie Brilliant Blue. The purification profiles from
transgenic cultures overexpressing AN3 is shown in FIG. 2. Protein bands
were cut, in-gel digested with trypsin and subjected to MALDI-TOF/TOF
mass spectrometry for protein identification. After substracting
background proteins, identified by the control purifications described in
example 2 and in other analyses (GUS and cytosolic GFP, Van Leene et al.,
2007), from the obtained hit list we identified 25 AN3 interacting
proteins (Table 2). These can be divided into two groups: 14 proteins
were confirmed experimentally and 11 proteins were identified only in one
out of four TAP experiments.

[0026] Among the experimentally confirmed AN3 interactors six proteins act
as subunits of macromolecular machines that remodel chromatin structure.
A database survey (ChromDB, Gendler et al., 2008) illustrates that all of
them belong to the SWI/SNF ATPase family. SWI/SNF chromatin remodeling
ATPases are conserved in the animal and the plant kingdom and regulate
transcriptional programs in response to endogenous and exogenous cues.
This suggests that the transcriptional activity of AN3 is regulated
through chromatin remodeling. In agreement, the human AN3 homolog SYT was
also shown to interact with the SWI/SNF complex components BRM and Brg1
(Thaete et al., 1999; Perani et al., 2003; Ishida et al., 2004).

[0028] With the exception of the SWI/SNF chromatin remodeling complex
subunits all other 19 identified AN3 interactors are not or poorly
characterized. Table 3 gives an overview of there GO biological process
and molecular function.

[0029] Among them four interactors (At4g16143, At1g09270, At3g06720 and
At5g53480) are involved in nucleocytoplasmic trafficking which identifies
AN3 as one of the targets of plant nuclear transporters. Indeed a precise
cellular localization is essential for protein function and nuclear
localization is a key to the function of transcription factors. In
plants, nucleocytoplasmic trafficking plays a critical role in various
biological processes (Meier, 2007; Xu & Meier, 2008) and nuclear
transporters have been shown to be involved in regulating different
signal transduction pathways during plant development (Bollman et al.,
2003) and in plant responses to biotic (Palma et al., 2005) and abiotic
stresses (Verslues et al., 2006).

[0030] Another AN3 interactor, that is yet not characterized, is the
trehalose phosphatase/synthase 4 (TPS4). Several studies in plants imply
an important role of trehalose biosynthesis for plant growth, development
and stress tolerance (Grennan, 2007). In the case of Arabidopsis TPS1,
knockout mutants display an embryo lethal phenotype, suggesting a role of
this gene in plant development (Eastmond et al., 2002). In addition,
overexpression of TPS1 shed light on its role as a regulator of glucose,
abscisic acid, and stress signalling (Avonce et al., 2004). The latter
study, together with a recent analysis of a rice TPS triggering abiotic
stress response gene induction when overexpressed (Ge et al., 2008),
suggests a possible role for TPS genes in regulating transcriptional
signaling pathways.